Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America.

Abstract

NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2) yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC) cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM) kinase activated by cisplatin and promote downstream gamma-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular-weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy.

Induction of caspases by exogenous expression of NPRL2 and cisplatin in non-small cell lung cancer cells.

(A) Induction of apoptosis in H1299 cells treated NPRL2 in the presence or absence of cisplatin (CDDP) by flow cytometry analysis with TUNEL staining. The PBS treated and empty vector (EV) treated cells were used as mock and negative controls, respectively. Error bars indicate SDs of the mean in three individual experiments (*, P<0.05; **, P<0.0005). (B), Activation of caspase cascades in H1299 cells by Western blot analysis. Several caspases in H1299 cells were examined by Western blotting 72 h after treatment with empty vector (EV) (with or without cisplatin) or with NPRL2 (with or without cisplatin). In cells treated with NPRL2 and cisplatin, caspases-3, -2, -8, and -9 and poly (ADP-ribose) polymerase (PARP) were cleaved. Also, caspase-2 was activated by treatment with only NPRL2.

(A), Effects of ectopic expression of NPRL2 on expression and phosphorylation of ATM and NBS1 proteins.H1229 cells treated with an empty vector (EV) vector and IC20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of ataxia telangiectasia mutated (ATM) kinase, phospho-ATM (Ser1981), NBS1, and phospho-NBS1 (Ser343). β-actin was used as a loading control. Increases in phospho-ATM and phospho-NBS1 were observed in cells treated with NPRL2 or NPRL2 and cisplatin in a time-dependent manner, but not in cells treated with the control empty vector and cisplatin. (B), Effects of NPRL2-specific siRNA (Si) on expression of NPRL2 and ATM proteins in the presence and absence of cisplatin (CDDP). The scrambled siRNA (Sc) was used as a non-specific control.

γ-H2AX, induced by cisplatin treatment, is also strongly enhanced by NPRL2.

H1229 cells treated with an empty vector and IC20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment. (A) Western blot shows remarkably enhanced expression of γ-H2AX in cells treated with NPRL2 and cisplatin compared with cells treated with empty vector and cisplatin. (B) γ-H2AX expression was examined at 48 h after treatment by immunofluorescence staining and flow cytometric analysis. γ-H2AX expression was detected in cells treated with empty vector and cisplatin or NPRL2 but not in cells treated with empty vector only, and this expression was dramatically enhanced in cells treated with NPRL2 and cisplatin. Magnification: ×800. Mean fluorescein isothiocyanate (FITC) intensity was also examined at 24, 48, and 72 h after treatment. At these three time points, γ-H2AX expression in cells treated with NPRL2 and cisplatin was significantly higher than that in cells in all other treatments (P<0.02 or P<0.0005). Bars, SDs of the mean in two individual experiments. (C) γ-H2AX expression was analyzed by immunofluorescence staining in an orthotopic model of H322 pleural dissemination, as described in the section. The pleural tumor cells from the mice treated with LacZ and cisplatin or NPRL2 were weakly stained; in contrast, γ-H2AX was hyperexpressed in cells treated with NPRL2+ cisplatin. Magnification: ×400.

Combination of NPRL2 and cisplatin increases the activation of both Chk1 and Chk2.

(A) H1229 cells treated with empty vector and IC20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC20 dose of cisplatin. (B) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. (C) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC20 dose of cisplatin, NPRL2 or NPRL2+ IC20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin (P<0.003). Chk2 kinase activity was more strongly enhanced in cells treated with NPRL2 or NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin (P<0.0001). Bars, SDs of the mean in four individual experiments.

NPRL2 enhances the effect of cisplatin that can activate cell cycle checkpoints.

H1229 cells treated with empty vector + IC20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2+ IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed by Western blotting for expression of cell cycle signaling molecules. Control H1299 cells were treated with empty vector and harvested at 72 h after treatment. β-actin was used as a loading control. (A) Cdc25A was degraded by the treatment of NPRL2 or IC20 dose of cisplatin 72 h later, and this degradation in treatment of NPRL2+ cisplatin strongly appeared 24 to 72 h later. P-Cdc25C was slightly increased by treatment with cisplatin; in contrast, it was remarkably increased by treatment with NPRL2 or NPRL2+ cisplatin. P-Cdc2 and P-SMC1 were clearly enhanced more with NPRL2+ cisplatin treatment than with cisplatin or NPRL2 treatment. (B) Immunoprecipitation Western blotting (IP-WB) analysis for protein-protein interaction between Cdc2-cyclin B1. H1299 cells were transfected with either empty vector or NPRL2 plasmid with or without IC20 value of cisplatin. The backbone plasmid vector without NPRL2 was used as a transfection control. Protein extracts were collected 72 h after transfection and immunoprecipitated with either anti-Cdc2 or anti-cyclin B1 antibody and immunoblotted with Cdc2 or cyclin B1 antibody. NPRL2 and cisplatin treatment remarkably degraded the interaction of Cdc2/cyclin B1 complex.

The cell cycle in tumor cells treated with NPRL2 and IC20 value of cisplatin was analyzed by flow cytometry with use of the APO-BRDU KIT. (A) The shift in cell cycle parameters of cells treated with NPRL2 is manifested as clear G1 and G2 peaks 72 h after treatment. In contrast, the cell cycle distribution showed an increase in the G2/M population after treatment with empty vector and cisplatin compared with cells treated with NPRL2 alone. The shift in cell cycle distribution was seen in cells treated with NPRL2+ cisplatin: the G2/M phase population strongly increased, whereas the G1 phase population decreased. (B) The cell cycle shift was analyzed at 24, 48, and 72 h after treatment. The combination of NPRL2 and cisplatin significantly increased the G2/M population in a time-dependent manner (P<0.001 at 48 h; P<0.0001 at 72 h) and increased the sub-G0-G1 population (P<0.001 at 72 h), compared with all other treatments. Bars, SDs of the mean from three individual experiments.